Method for extracorporeal removal of a pathogenic microbe, an inflammatory cell or an inflammatory protein from blood

ABSTRACT

The present invention relates to a method for extracorporeal removal of a pathogenic microbe, an inflammatory cell or an inflammatory protein from mammalian blood/use of a device comprising a carbohydrate immobilized on a solid substrate, said carbohydrate having a binding affinity for a pathogenic microbe, an inflammatory cell or an inflammatory protein, for extracorporeal removal of said pathogenic microbe, inflammatory cell or inflammatory protein from mammalian blood/use of a carbohydrate having a binding affinity for a pathogenic microbe, an inflammatory cell or an inflammatory protein, wherein said carbohydrate is immobilized on a solid substrate, in the preparation of a device for treatment of a condition caused or aggravated by said pathogenic microbe, inflammatory cell or inflammatory protein and a method for treatment of a mammalian subject suffering from a condition caused or aggravated by a pathogenic microbe, an inflammatory cell or an inflammatory protein.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 15/678,580, filed Aug. 16, 2017, allowed, which isa continuation application of U.S. patent application Ser. No.14/873,133, filed Oct. 1, 2015, now U.S. Pat. No. 9,764,077, which is acontinuation application of U.S. patent application Ser. No. 12/086,126,filed Sep. 10, 2008, now U.S. Pat. No. 9,173,989, which application is a35 U.S.C. § 371 National Phase Application of PCT/SE2006/001421, filedDec. 13, 2006, which application claims priority to SE 0502750-3, filedDec. 13, 2005, the disclosures of which are hereby incorporated byreference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention relates to a method for extracorporeal removal ofa pathogenic microbe, an inflammatory cell or an inflammatory proteinfrom mammalian blood; use of a device comprising a carbohydrateimmobilized on a solid substrate, said carbohydrate having a bindingaffinity for a pathogenic microbe, an inflammatory cell or aninflammatory protein, for extracorporeal removal of said pathogenicmicrobe, inflammatory cell or inflammatory protein from mammalian blood;use of a carbohydrate having a binding affinity for a pathogenicmicrobe, an inflammatory cell or an inflammatory protein, wherein saidcarbohydrate is immobilized on a solid substrate, in the preparation ofa device for treatment of a condition caused or aggravated by saidpathogenic microbe, inflammatory cell or inflammatory protein; and amethod for treatment of a mammalian subject suffering from a conditioncaused or aggravated by a pathogenic microbe, an inflammatory cell or aninflammatory protein.

BACKGROUND Biology

During a long evolution, many pathogenic microorganisms have learned toexploit eukaryotic cell surface glycoconjugates, i.e. glycolipids,glycoproteins and proteoglycans, as receptor molecules for cellattachment to facilitate tissue colonization and invasion processes. Inbrief, specific proteins called adhesins of the surface of bacteria,viruses, fungi and parasites interact with carbohydrate chains ofglycoconjugates which enable microbes to colonize mucosal surfaces andtissue lesions.

The role of sialic acid in binding of pathogens to host cells has beenreported over many years. Only recently proteoglycans with theircarbohydrate chains (glycosaminoglycans) were shown to bind manydifferent pathogens. By removing terminal carbohydrate moieties of thesevarious glycoconjugates with sialidase and other exoglycosidases or withglycosaminoglycan (GAG) degrading enzymes on the cells in monolayers,these structures were proven to be receptor molecules for varioussialoadhesins and heparan sulfate binding proteins (HeBPs).

These mechanisms are summarized in a review article by Siiri Hirmo,Meeme Utt and Torkel Wadström, Biology, Biochemistry, ClinicalBiochemistry, Volume 12, including Proceedings from the 17thInternational Lectin Meeting in Würzburg, 1997, edited by Edilbert vanDriessche, Sonia Beeckmans and Thorkild C. Bog-Hansen, published byTEXTOP, Lemchesvej 11, DK-2900 Hellerup, Denmark, ISBN number87-984583-0-2.

During microbial infections, inflammatory mediators are released andactivated. These so-called “pro-inflammatory cytokines” include tumornecrosis factor alpha and beta (TNF-α and TNF-β), interleukin-1 (IL-1),and interleukin-6 (IL-6). These cytokines are part of the inflammatoryresponse of sepsis. Multiple organ failure induced by sepsis iscurrently the leading cause of death in intensive care units.

In connection with microbial infections and cardiovascular surgery, forinstance cardiopulmonary bypass, inflammatory responses are elicited andhave a multitude of biological consequences, ranging from subclinicalorgan dysfunction to severe multiorgan failure. Cytokines are thought tobe important mediators in this response.

The cytokines mentioned above have a capacity to bind selectively to arange of glycosaminoglycans, or GAGs, including heparan sulfate intissues and on the surface of both endothelial cells and leucocytes.

Receptors

Heparan sulfate is a glycosaminoglycan that is present on the surface ofalmost all mammalian cells. It is built up by alternating D-glucosamineand uronic acid residues (L-iduronic and D-glucuronic). Heparan sulfatesare highly charged (sulfated) heterogeneous polysaccharides andrepresent the carbohydrate portion of many glycoconjugates (syndecan,perlecan, glypican) on the cell surface.

Many microbes utilize heparan sulfates on the surface of the mammaliancell as receptors. This mechanism is general and valid for almost allbacteria, virus and parasites. Some microorganisms utilize more than oneglycoconjugate receptor. Examples of other receptors that are usedtogether with heparan sulfate are specific chondroitin sulfates andsialic acid containing glycoproteins.

Heparan sulfate/chondroitin sulfate binding microbes are exemplified byviruses like herpes simplex virus type 1 (HSV-1), causative agent oforolabial herpes; herpes simplex virus type 2 (HSV-2), causative agentof genital herpes; cytomegalovirus (CMV), the major complicating agentin immunosuppressed patients; dengue virus, which causes recurrentfevers; and human immunodeficiency virus (HIV); and by bacteria likeHelicobacter pylori, Streptococcus sanguis, Streptococcus mutans,Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, andMycobacterium tuberculosis; and parasites like Plasmodium falciparum(which causes malaria), and Trypanosoma cruzi (which causestrypanosomiasis).

Further, cytokines, like TNF-β, also utilize heparan sulfate on cellsurfaces for binding and activation.

Heparin as a Receptor

Heparin is a polysaccharide, which is isolated from mammalian tissue.Since its discovery in 1916 by the American scientist McLean, heparinhas been recognized for its blood anticoagulant properties and heparinhas, for more than 50 years, been used clinically as a bloodanticoagulant and antithrombotic agent.

Whereas heparan sulfates are ubiquitous components of alltissue-organized animal life forms, heparin has a very particulardistribution in mammalian tissue. Heparin is, in contrast to the heparansulfates, present only in the basophilic granules of mast cells.However, today, in addition to its established place in prevention andtherapy of thromboembolic disorders, heparin has demonstrated a broadspectrum of different activities independent of anticoagulation.

A large number of proteins in blood bind, with high affinity, to heparinand/or heparan sulfate. Examples are antithrombin (AT), fibronectin,vitronectin, growth factors (e.g. the fibroblast growth factors, theinsulin like growth factors etc). Human serum albumin (HSA) also binds,but with a lower affinity. On the other hand, HSA is present in largeamounts in blood.

To utilize these properties of heparin for hindering infections,introducing heparin fragments and/or sialic containing fragments intothe vascular system has been contemplated. Thereby, it was thought,these fragments would bind to the lectins on the microbes, block themand thus hinder them from binding to the receptors on the mammalian cellsurface. This concept has been tried by many scientists but with limitedsuccess, in most cases due to bleeding complications when large amountsof heparin are introduced into the vascular system.

U.S. Pat. No. 6,197,568 dicloses methods for isolation and detection offlaviviruses and other hemorrhagic fever viruses, such as dengue virus,based on the sulfated polyanion-dependent interaction of flavivirusesand hemorrhagic fever viruses.

Extracorporeal devices are used in a variety of clinical situationsincluding kidney dialysis, cardiopulmonary bypass and plasmapheresis.

“Extracorporeal therapies” means procedures in which desired productslike oxygen, blood-anticoagulants, anesthetics etc can be added to bodyfluids. Conversely, undesired products like toxins etc can be removedfrom body fluids outside the body. Examples are haemodialysis andhaemofiltration which represent technologies whereby blood is rinsedfrom waste products.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for treatmentof a mammal suffering from diseases or conditions caused or aggravatedby different pathogenic microbes, inflammatory cells or inflammatoryproteins by removal of said pathogenic microbes, inflammatory cells orinflammatory proteins from the blood of said mammal.

Another object of the present invention is to provide a method forextracorporeal removal of pathogenic microbes, inflammatory cells orinflammatory proteins from mammalian blood.

The above mentioned objects, as well as further objects of theinvention, which should be apparent to a person skilled in the art afterhaving studied the description below, are accomplished by the differentaspects of the present invention as described herein.

A first aspect of the present invention provides a method forextracorporeal removal of a pathogenic microbe, an inflammatory cell oran inflammatory protein from mammalian blood, comprising the steps:

-   -   a) providing a sample of mammalian blood,    -   b) bringing said sample into contact with a carbohydrate        immobilized on a solid substrate, said carbohydrate having a        binding affinity for a pathogenic microbe, inflammatory cell or        inflammatory protein, under conditions allowing binding of any        pathogenic microbes, inflammatory cells and inflammatory        proteins in said blood sample to the carbohydrate,    -   c) separating the sample from the solid substrate, such that        said pathogenic microbe, inflammatory cell or inflammatory        protein is at least partially retained on the solid substrate,        and    -   d) recovering said sample containing a reduced amount of said        pathogenic microbe, inflammatory cell or inflammatory protein.

The prior-art concept of introducing heparin fragments and/or sialiccontaining fragments into the vascular system of patients has shownlimited success, in most cases due to bleeding complications when largeamounts of heparin are introduced into the vascular system. The methodaccording to the invention circumvents these problems by thecarbohydrates being immobilized on a solid surface, as is inter aliadescribed in Preparatory example 6.

Use of immobilized carbohydrates as defined by the method according tothe invention also provides a further unexpected advantage. Theinventors have found that the carbohydrate has to be immobilized onto asolid surface to have the capacity that is necessary for binding asignificant amount of the compounds that are to be removed. Thisunexpected property is described in Comparative example 1 and Example 1for HSV-1, showing that in solution, less than 3% of the virus binds toan excess of heparin (Comparative example 1) while more than 94% of thevirus binds to immobilized heparin (Example 1).

The method of the present invention enables safe and efficient treatmentof patients suffering from sepsis or septic shock by removing pathogenscausative of the condition from the patient's blood stream. The methodof the present invention allows removal of many different pathogenicmicrobes, inflammatory cells and inflammatory proteins. Examples ofpathogenic microbes commonly associated with sepsis that may be removedusing the method of the invention include staphylococci, such asStaphylococcus aureus, streptococci and E. coli.

As both heparin and heparan sulfates bind to a large number ofcomponents, as exemplified in the background section, it was expectedthat a heparin surface would be covered with many of these proteins,when brought into contact with blood, thus preventing the microbes fromattaching. The present inventors have surprisingly found that highlyefficient purification of serum and whole blood from mammals, includinghumans, can be achieved using a method and a device according to thepresent invention. It is disclosed herein that a column of moderate sizemanaged to almost completely remove considerable amounts of viruses fromblood serum and whole blood. See e.g. Examples 2, 3 and 4.

In an embodiment, said pathogenic microbe is selected from the groupconsisting of bacteria, viruses and parasites.

In an embodiment, said pathogenic microbe is a virus. In a more specificembodiment, said virus is selected from the group consisting of herpessimplex virus type 1, herpes simplex virus type 2, Influenza A virus,cytomegalovirus and human immunodeficiency virus. In another morespecific embodiment, said virus is selected from the group consisting ofherpes simplex virus type 1 or herpes simplex virus type 2.

In another embodiment, said pathogenic microbe is a bacterium. In a morespecific embodiment, said bacterium is selected from the groupconsisting of Helicobacter pylori, Streptococcus sanguis, Streptococcusmutans, Staphylococcus aureus, Escherichia coli, Pseudomonas aureginosaand Mycobacterium tuberculosis. In a preferred embodiment, saidpathogenic microbe is Helicobacter pylori or Staphylococcus aureus.

In yet another embodiment, said pathogenic microbe is a parasite. In amore specific embodiment, said parasite is selected from the groupconsisting of Plasmodium falciparum and Trypanosoma cruzi.

In a further embodiment, said inflammatory cell is selected from thegroup consisting of inflammatory lymphocytes and inflammatorymacrophages.

In yet a further embodiment, said inflammatory protein is apro-inflammatory cytokine. In a more specific embodiment, saidpro-inflammatory cytokine is selected from the group consisting of tumornecrosis factor alpha (TNF-α), tumor necrosis factor beta (TNF-β),interleukin-1 (IL-1), and interleukin-6 (IL-6).

In an embodiment, said mammalian blood is human blood.

In an embodiment of the inventive method, said carbohydrate is selectedfrom the group consisting of heparin, heparan sulfate, chondroitinsulfate, carbohydrates comprising sialic acid and carbohydratescomprising neuramic acid. In a more specific embodiment, saidcarbohydrate is heparin.

In yet another embodiment, said solid substrate comprises microparticlesor hollow fibres. In certain embodiments of the invention, the materialof said solid substrate is selected from the group consisting of glass,cellulose, cellulose acetate, chitin, chitosan, crosslinked dextran,crosslinked agarose, polypropylene, polyethylene, polysulfone,polyacrylonitrile, silicone, Teflon and polyurethanes.

In a further embodiment, said carbohydrate is covalently linked to saidsolid substrate. In a more specific embodiment, said carbohydrate islinked to said solid substrate by covalent end-point attachment.Covalent attachment of a carbohydrate to a solid substrate providesbetter control of parameters such as surface density and orientation ofthe immobilized molecules as compared to non-covalent attachment. Theseparameters have been shown by the inventors to be important in order toprovide optimal pathogen binding to the immobilized carbohydratemolecules. The surface concentration of the carbohydrate on the solidsubstrate should preferably be in the range of 1-10 μg/cm². Covalentend-point attachment means that the carbohydrate is covalently attachedto the solid substrate via the terminal residue of the carbohydratemolecule. A second aspect of the present invention provides use of adevice comprising a carbohydrate immobilized on a solid substrate, saidcarbohydrate having a binding affinity for a pathogenic microbe, aninflammatory cell or an inflammatory protein, for extracorporeal removalof a pathogenic microbe, inflammatory cell or inflammatory protein frommammalian blood.

Embodiments of a use according to the second aspect of the inventioncorrespond to those specified above for the method according to thefirst aspect of the present invention regarding the pathogenic microbe,inflammatory cell, inflammatory protein, mammalian blood, carbohydrate,solid substrate and immobilization.

A third aspect of the invention provides use of a carbohydrate having abinding affinity for a pathogenic microbe, an inflammatory cell or aninflammatory protein, wherein said carbohydrate is immobilized on asolid substrate, in the preparation of a device for treatment of acondition caused or aggravated by a pathogenic microbe, inflammatorycell or inflammatory protein.

Embodiments of a use according to the third aspect of the inventioncorrespond to those specified above for the method according to thefirst aspect of the present invention regarding the pathogenic microbe,inflammatory cell, inflammatory protein, mammalian blood, carbohydrate,solid substrate and immobilization.

A device as referred to in the use and method according to the inventionmay comprise a conventional device for extracorporeal treatment of bloodand serum from patients, e.g. suffering from renal failure.

Local blood flow patterns in blood contacting medical devices forextracorporeal circulation are known to influence clot formation viashear activation and aggregation of platelets in stagnant zones.Consequently, a device as used in the second, third and fourth aspectsof the invention should be designed in a fashion that does not createthese problems.

A device as used in some embodiments of the invention may for examplehave the following properties:

-   -   A blood flow in the range of 1-500 ml/min, preferably 5-250        ml/min.    -   Low flow resistance.    -   Large surface area of substrate having carbohydrates immobilized        thereto, e.g. about 0.1-1 m².    -   Stable coating (no leakage of carbohydrate to the blood in        contact therewith).    -   Proper haemodynamic properties in the device (no stagnant        zones).    -   Optimal biocompatibility.

A non-limiting example of such a device, which can be used in a use or amethod according to the present invention, is a pediatric haemoflowdialyzer such as the Prisma M10 haemofilter/dialyzer from Gambro AB,Sweden. Other models or types of devices for extracorporeal treatment ofblood or serum may also be used.

A fourth aspect of the present invention provides a method for treatmentof a mammalian subject suffering from a condition caused or aggravatedby a pathogenic microbe, an inflammatory cell or an inflammatoryprotein, comprising the steps:

-   -   a) extracting blood from the subject,    -   b) bringing the extracted blood into contact with a device        comprising a carbohydrate immobilized on a solid substrate, said        carbohydrate having a binding affinity for a pathogenic microbe,        an inflammatory cell or an inflammatory protein, under        conditions allowing binding of a pathogenic microbe, an        inflammatory cell or an inflammatory protein to the        carbohydrate, and    -   c) reintroducing the blood, containing a reduced amount of said        pathogenic microbe, inflammatory cell or inflammatory protein,        into the blood-stream of the subject.

In an embodiment of the treatment method according to the presentinvention, the extraction and reintroduction of blood is performed in acontinuous loop, which loop comprises a part of the bloodstream of thesubject.

Embodiments of a method for treatment according to the fourth aspect ofthe invention correspond to those specified above for the methodaccording to the first aspect of the present invention regarding thepathogenic microbe, inflammatory cell, inflammatory protein, mammalianblood, carbohydrate, solid substrate and immobilization.

As used herein, the term “pathogenic microbe” means a microbe, which cancause disease in a living organism when introduced into said organism.Examples of “pathogenic microbes” include bacteria, viruses andparasites.

As used herein, the term “inflammatory cell” means a cell, which isinvolved in inflammatory response in a mammal. Examples of “inflammatorycells” include inflammatory lymphocytes and inflammatory macrophages.

As used herein, the term “inflammatory protein” means a protein, such asa cytokine, released for instance in connection with microbial infectionor immunization.

As used herein, the term “cytokine” means a protein, released forinstance in connection with microbial infection or immunization,selected from the group consisting of interleukins, interferons,chemokines and tumour necrosis factors.

EXAMPLES Preparatory Example 1 Amination of Sephadex G 25

Sodium metaperiodate (NaIO₄, 6.0 g) was dissolved in water (994 ml) andadded to Sephadex G 25 (Pharmacia Biotech, Uppsala, Sweden) (50 g) in 1l water. The mixture was kept in the dark under shaking for 24 h. Afterfiltration and washing with water 5×1 l and finally 0.1 M phosphatebuffer, pH 7.0, the resulting product was suspended in phosphate buffer,pH 7.0 (350 ml) and a solution of polyethylenimine (100 ml Lupasol(BASF, Germany), 5% in water) was added. The gel was stabilized byaddition of an aqueous solution of NaBH₃CN, sodium cyanoborohydride (0.5g in 100 ml, phosphate buffer, 0.1 M, pH 7.0). The gel was filtered andwashed as described above and finally washed with acetate buffer (500ml, 0.1 M, pH 4.0), yielding aminated Sephadex G 25 (85 g).

Preparatory Example 2

Covalent End-Point Attachment of Heparin onto a Chromatographic Gel

Aminated Sephadex G 25 (85 g) obtained as described in Preparatoryexample 1 was suspended in acetate buffer (800 ml, 0.1 M, pH 4.0) and4.0 g nitrous acid degraded heparin (heparin from Pharmacia, Sweden) wasadded. After shaking for 0.5 h, NaBH₃CN (0.4 g) was added. The reactionmixture was shaken for 24 h and then processed as above, yieldingheparinized Sephadex G 25 (80 g).

The gel contains 2% heparin (w/w, sulfur analysis). The Sephadex G 25beads have an average diameter of 50-150 μm. A rough calculation revealsthat 1 cm³ contains 10⁶ beads which gives a bead surface area of 0.03m²/cm³. Further, if heparin is attached only to the surface of thebeads, a heparinized Sephadex G 25 with 2% heparin w/w has about 0.003μg heparin/cm².

Preparatory Example 3

Covalent Attachment of Heparin onto Aminated Glass Wool

A glass wool material is heparinized using the general proceduredescribed below.

Glass wool is thoroughly cleaned with acid (HCl), rinsed with absoluteethanol, and dried in an oven at 100° C. for 4 hours.

Reactive amino functions are introduced on the glass wool surface bytreatment with an aqueous solution of polyamine, polyethylenimine (PEI)or chitosan. For some purposes, the polyamines may be stabilized on thesurface by crosslinking with bifunctional reagents, such ascrotonaldehyde or glutaraldehyde.

The coating is further stabilized by ionic cross linking with a sulfatedpolysaccharide (dextran sulfate or heparin). If necessary, these stepsare repeated and a sandwich structure is built up. Careful rinsing(water, suitable buffers) should be performed between each step. After alast addition of PEI or chitosan, end-point attachment (EPA) to theaminated surface of native heparin is done by reductive amination,utilizing the aldehyde function in the reducing terminal residue innative heparin. The coupling is performed in aqueous solution, byreductive amination (cyanoborohydride, CNBH₃ ⁻) essentially as describedin Preparatory example 2.

Surface analysis as described in Preparatory example 2 reveals thatapproximately 10 mg/cm² of heparin is coupled to the glass surface.

Preparatory Example 4

Covalent Attachment of Heparin onto Aminated Polymeric Surfaces

A polymeric surface was heparinized using the general proceduredescribed below.

The polymeric surface is etched with a oxidizing agent (potassiumpermanganate, ammoniumperoxidisulfate) in order to introduce hydrophiliccharacteristics together with some reactive functional groups (—SO₃H,—OH, —C═O, —C═C—). The surface can also be etched with plasma or corona.

Reactive amino functions are introduced by treatment with a polyamine,polyethylenimine (PET) or chitosan. For some purposes the polyamines maybe stabilized on the surface by cross linking with bifunctionalreagents, such as crotonaldehyde or glutaraldehyde.

The coating is further stabilized by ionic cross linking with a sulfatedpolysaccharide (dextran sulfate or heparin). If necessary these stepsare repeated and a sandwich structure is built up. Careful rinsing(water, suitable buffers) should be performed between each step. After alast addition of PEI or chitosan, end-point attachment (EPA) to theaminated surface of native heparin is done by reductive amination,utilizing the aldehyde function in the reducing terminal residue innative heparin. A more reactive aldehyde function in the reducingterminal residue can be achieved by partial, nitrous degradation ofheparin. This shortens the reaction time, but the immobilized heparinwill have a lower molecular weight. The coupling is performed in aqueoussolution, by reductive amination (cyanoborohydride, CNBH₃ ⁻) essentiallyas described in Preparatory example 2.

1-10 μg/cm² of heparin can be coupled to all hydrophilic surfaces likeglass, cellulose, chitin etc, and more or less all hydrophobic polymerslike polyvinyl chloride, polyethylene, polycarbonate, polystyrene, PTFEetc.

Preparatory Example 5

Covalent Single- or Multipoint Attachment of Heparin onto PolymericSurfaces

Performed as described in Preparatory example 2, with the exception thatthe aldehyde functions were introduced in the heparin chain by oxidationwith sodium periodate in aqueous solution.

Preparatory Example 6

Attachment of Heparin onto the Inner Lumen of Hollow Fibers

In this preparatory example, a pediatric haemoflow dialyzer was used.The fibers of the dialyzer were made of polysulfone with an innerdiameter of 200 microns and a wall thickness of 40 microns. The totalsurface area of the blood contacting material was 4000 cm² and thepriming volume was 28 ml.

The amination procedure was performed as generally described inPreparatory example 4 with the exception that the etching step wasomitted. Polysulfone is hydrophilic and does not need etching.Immobilization of heparin was performed by pumping a solution containingnitrous acid degraded heparin (heparin from Pharmacia) together withNaBH₃CN as described in Preparatory example 2. As measurement of theamount of heparin is a destructive procedure, a reference dialyzer thatwas heparinized under identical conditions was sacrificed and its fibersare subjected to sulfur analysis. The results revealed a heparin contentof about 5 μg heparin/cm², which corresponds to a content of 20 mgheparin in the device.

Preparatory Example 7

Covalent Attachment of Oligomers with Terminal Sialic Acid Residues ontothe Inner Lumen of Hollow Fibers

In this preparatory example, the aldehyde group at the reducing terminalresidue was used for coupling. Amination of the fibers was performed asdescribed in Preparatory example 6 and coupling of the oligosaccharideof formula 1, which contains terminal sialic acid residues, wasperformed by circulating the compound of formula 1, dissolved in acetatebuffer (800 ml, 0.1 M, pH 4.0) together with NaBH₃CN (0.4 g), at roomtemperature for 24 h. The results revealed a sialic acid content of ca.2 μg/cm².

Comparative Example 1 Binding of HSV-1 to Heparin in Solution

A solution (10 μl) containing 10⁷ plaque forming units of virus (Herpessimplex virus type 1 strain KOS321) was incubated with 20 μl of³H-labelled heparan sulfate (HS) in a total volume of 400 μl of bufferedNaCl for 30 min at 37° C. Thereafter, the solution was centrifugedthrough a Microsep 1 M filter, retaining virus and bound HS. 2.3% of HSwas bound (479 CPM) to the virus, while 97.7% of the HS was unbound andpassed through the filter.

Example 1

Removal of HSV-1 and HSV-2 Virus Particles from Buffered Saline byBinding to Heparin Immobilized on Sephadex Beads

Sephadex beads coated with heparin, as in Preparatory example 2, weresoaked in buffered NaCl (PBS) and 0.8 ml was transferred to each of twosmall disposable columns, forming a gel layer of approximately 1 cm.After washing three times, 50 μl of ³H-thymidine radiolabelled viruseswere suspended in 150 μl of PBS. 10⁹ plaque forming units of HSV-1,corresponding to 10¹¹ virus particles, were added to column 1, and 10⁸plaque forming units of HSV-2, corresponding to 10¹⁰ virus particles,were added to column 2. Virus was allowed to adsorb to the respectivecolumns. Thereafter, 0.8 ml of PBS was added to each column and thepass-through fluid was collected for estimation of unabsorbed virus.

Subsequently, both columns were washed 4 times with 1 ml of PBS, and thewashings were collected as fractions for quantification of washed outvirus. These, and the following fractions, were transferred toscintillation vials and quantified with regard to amount of virusthrough determination of cpm in a beta counter. In the next step, thecolumns were subjected to elution of the respective heparin-boundviruses three times by 1 ml of 2 M NaCl, and the three fractions werecollected from each column. Following that, elution was performed bytwice adding 1 ml of 5% SDS in PBS (PBS-SDS), and the two fractions fromeach column were collected. Finally, the heparin-coated beads from thetwo columns were each suspended in 1 ml of PBS-SDS, and 200 μl aliquotswere subjected to quantification of remaining bound virus particles bydetermination of radioactivity.

The results are shown in Table 1 below. As shown, only 5.5% of HSV-1particles and 11.7% of HSV-2 particles did not adsorb to the column.Moreover, since the viral DNA and not their heparin-binding proteins arelabeled with radioactivity, these non-adsorbed particles might representnon-infectious viruses with disrupted envelopes (i.e. the outer,fragile, parts of the virus that bind to heparin). The rest of theviruses (94.5% for HSV-1 and 88.3% of HSV-2) bind to the heparin-coatedbeads in the column. The binding appears to be strong, judging from thefact that only 0.5% of HSV-1 and 1.1% of HSV-2 was removed by 4successive washings. The limited ability of 2 M NaCl at 3 successiveattempts to elute the viruses underscores the high-affinitycharacteristic of the binding of both viruses to the heparin-coatedbeads. In contrast, substantial quantities of HSV-1 and HSV-2 wereeluted by PBS-SDS.

A total of 48% of HSV-1 and 68.8% of HSV-2 were recovered from thecolumns. This can be attributed to the fact that 2 M NaCl spontaneouslydecreased the radioactivity of the samples by approximately 30%according to our past observations, and that SDS-PBS probably also hasthis effect.

Taken together, the results prove the principle that HSV-1 and HSV-2virus particles can be removed from a fluid phase by passage through ashort column containing heparin-coated Sephadex beads, and thatextracted viruses bind with high affinity to the columns.

TABLE 1 Binding of radiolabelled HSV virus particles, suspended inbuffered NaCl, to heparin-Sephadex beads. Binding of HSV to heparincolumn (% of input virus = control = 100%) HSV-1 HSV-2 Input virus(control) 100.0 100.0 Unadsorbed virus 5.5 11.7 Washed from column1^(st) washing 0.2 0.4 2^(nd) washing 0.1 0.3 3^(rd) washing 0.1 0.24^(th) washing 0.1 0.2 total unadsorbed + washed 6.0 12.8 Eluted with 2MNaCl 1^(st) elution 7.8 14.7 2^(nd) elution 1.6 1.9 3^(rd) elution 0.11.3 Eluted with 5% SDS 1^(st) elution 10.6 11.9 2^(nd) elution 18.5 18.3total eluted NaCl + SDS 38.6 48.1 Uneluted from beads 4.1 7.9 totaluneluted 4.1 7.9 Total recovered 48.7 68.8 (unadsorbed + washed +eluted + uneluted)

Example 2

Removal of HSV-1 Virus Particles from Human Serum by Binding to HeparinImmobilized on Sephadex Beads

The experimental procedure as described in Example 1 was utilized withthe difference that the radiolabelled HSV-1 virus particles at aquantity of 10⁹ PFU equivalent to 10¹¹ virus particles were mixed with0.5 ml of human serum and then applied on heparin-coated beads in adisposable column. Thereafter, the procedure including elution andwashing was followed as in Example 1. The results are shown in Table 2below.

TABLE 2 Binding of radiolabelled HSV-1 virus particles (10¹¹), suspendedin human serum, to heparin-Sephadex beads (1 cm³). Binding of HSV-1 toheparin column (% of input virus = control = 100%) Input virus (control)100.0 Unabsorbed virus 2.4 Washed from column 1^(st) washing 0.9 2^(nd)washing 0.2 3^(rd) washing 0.1 4^(th) washing 0.2 total unabsorbed +washed 3.8 Eluted with 2M NaCl 1^(st) elution 2.1 2^(nd) elution 0.43^(rd) elution 0.2 Eluted with 5% SDS 1^(st) elution 3.5 total elutedNaCl + SDS 6.2 Total recovered 10.0 (unabsorbed + washed + eluted +uneluted) Remained on beads 90.0

As shown, 97.6% of the HSV-1 particles suspended in human serum werebound to the column. By washing 4 times, only 3.8 of the virus particleswere removed. Using 2 M NaCl, only 2.7% of the virus was eluted, and anadditional 3.5% were eluted by SDS. The conclusion of these results isthat suspending virus in serum, which is the real life situation duringsevere, disseminated infection, improved the performance of thevirus-removing column as regards binding of radiolabelled virus, andthat only 2.4% of the HSV-1 particles were unadsorbed. As a probableexplanation, serum proteins helped to stabilize the virus particles andthereby improved the removal of HSV-1 by the heparinized Sephadex beads.

Example 3

Removal of HSV-1 Virus Particles from Human Serum by Binding to HeparinImmobilized on a Hollow Fiber Haemoflow Dialyzer

The experimental procedure as described in Example 1 was utilized withthe difference that the radiolabelled HSV-1 virus particles at aquantity of 10⁹ PFU equivalent to 10¹¹ virus particles were mixed with0.5 ml of human serum and then applied on the heparin-coated hollowfiber dialyzer of Preparatory example 6. Thereafter, the procedureincluding elution and washing was followed as in Example 1. The resultsare shown in Table 3 below.

As shown, 92.3% of the HSV-1 particles suspended in human serum werebound to the column. By washing 4 times, only 4.2% of the virusparticles were removed. By 2 M NaCl, only 4.0% of the virus was eluted,and an additional 4.5% were eluted by SDS. The conclusion of theseresults is that the binding of virus particles suspended in human serumto heparinized fibers is comparable to that of similarly suspended virusbinding to heparin-coated Sephadex beads, and that only 7.7% of theHSV-1 particles were unabsorbed.

TABLE 3 Binding of radiolabelled HSV-1 virus particles, suspended inhuman serum, to heparinized hollow fibers. Binding of HSV-1 to heparincolumn (% of input virus = control = 100%) Input virus (control) 100.0Unabsorbed virus 7.7 Washed from column 1^(st) washing 3.1 2^(nd)washing 0.8 3^(rd) washing 0.1 4^(th) washing 0.2 total unadsorbed +washed 4.2 Eluted with 2M NaCl 1^(st) elution 3.3 2^(nd) elution 0.53^(rd) elution 0.2 Eluted with 5% SDS 1^(st) elution 4.5 total elutedNaCl + SDS 8.5 Total recovered 12.7 (unabsorbed + washed + eluted +uneluted) Remained on beads 87.3

Example 4

Removal of HSV-1 and HSV-2 Virus Particles from Human Whole Blood byBinding to Heparin Immobilized on Sephadex Beads

The experimental procedure as described in Example 1 was utilized withthe difference that the radiolabelled HSV-1 and HSV-2 virus particles ata quantity of equivalent to 10¹¹ virus particles per ml were mixed with1 ml of human blood and then applied to 1 ml heparin-coated beads in adisposable column. Thereafter, the procedure including elution andwashing was followed as in Example 1.

The results are shown in Table 4 below. As shown, 99.1% of the HSV-1particles and 99.8% of the HSV-2 particles suspended in human blood werebound to the column.

TABLE 4 Binding of radiolabelled HSV virus particles, suspended in humanblood, to heparin-Sephadex beads (1 cm³). Binding of whole blood HSV toheparin column (% of input virus = control = 100%) HSV-1 HSV-2 % % Inputvirus (control) 100.0 100.0 Unadsorbed virus 0.9 0.2

Example 5

Removal of Influenza a Virus from Human Serum by Binding to ImmobilizedOligosaccharides Containing Sialic Acid

Virus stocks of Influenza A H1N1 were replicated in MDCK cells grown at35° C. under standard conditions for 3 days, after which the cells werehomogenized and titrated to assess the number of focus-forming units(FFU)/ml. Virus particles were suspended in human serum to a finalconcentration of 10⁶ FFU/ml. A 10 ml suspension was applied on a sialicacid-coated hollow fiber dialyzer, prepared using the method describedin Preparatory example 7. After titrating the infectivity of theInfluenza A-containing serum after passage through the dialyzer andcomparing it with titers of an aliquot of the same virus-containingserum not passed through the device it was concluded that 87% of theInfluenza A virus FFU remained bound to the fibers.

Example 6

Removal of Helicobacter pylori and Staphylococcus aureus by Binding toImmobilized Heparin

Four sterile pipettes were packed with glass wool (0.5 ml) that washeparinized as described in Preparatory example 3. The “columns” thusformed were washed with 3 ml of sterile phosphate saline buffer (PBS),pH 7.2. Two different strains of H. pylori and two different strains ofS. aureus were tested. Each of the four different bacteria samples,suspended in PBS buffer, were applied to a separate “column”. Theamounts of bacteria in the samples were measured before application tothe column and after elution from the column by optical density (OD) at560 nm and viable counts (CFU/ml). As is evident from the table below,roughly 90% of H. pylori and roughly 50% of S, aureus bacteria wereimmobilized on the columns.

TABLE 6 Binding of H. pylori and S. aureus to heparinized glass wool.OD₅₆₀ CFU/ml Binding Bacteria In Out In Out % H. Pylori ATCC 8.33 0.81 2 × 10⁸ 1.5 × 10⁷ ~90 43504 H. Pylori ATCC 8.33 1.86  2 × 10⁸  1 × 10⁷~90 43504 S. aureus CCUG 9.30 5.5 7.7 × 10⁹ 3.6 × 10⁹ ~50 12600 S.aureus CCUG 9.30 6.9 7.7 × 10⁹ 3.6 × 10⁹ ~50 12600

What is claimed is:
 1. A method for treating a subject in need thereof,by extracorporeal removal of a pathogenic microbe, said methodcomprising: a) contacting said subject's whole blood with heparinimmobilized on a solid substrate, wherein heparin immobilizationconsists of a single covalent link of said terminal residue to saidsolid substrate by covalent end-point attachment, under conditionsallowing binding of said pathogenic microbe in said subject's wholeblood sample to the heparin; b) separating the whole blood from thesolid substrate; c) recovering said whole blood containing a reducedamount of said pathogenic microbe; and d) reintroducing into saidsubject said whole blood containing a reduced amount of said pathogenicmicrobe.
 2. The method of claim 1, wherein said pathogenic microbe is aparasite.
 3. The method of claim 2, wherein said parasite is selectedfrom the group consisting of Plasmodium falciparum and Trypanosomacruzi.
 4. The method of claim 1, wherein said solid substrate comprisesmicroparticles.
 5. The method of claim 1, wherein said solid substratecomprises fibers.
 6. The method of claim 1, wherein the material of saidsolid substrate is at least one member selected from the groupconsisting of glass, cellulose, cellulose acetate, chitin, chitosan,crosslinked dextran, crosslinked agarose, polypropylene, polyethylene,polysulfone, polyacrylonitrile, silicone, Teflon and polyurethanes.